Advancing <sup>89</sup>Zr-immuno-PET in neuroscience with a bispecific anti-amyloid-beta monoclonal antibody - The choice of chelator is essential
Authors: Thomas E. Wuensche, Natascha Stergiou, Iris Mes, Mariska Verlaan, Maxime Schreurs, Esther J. M. Kooijman, Bart Janssen, Albert D. Windhorst, Allan Jensen, Ayodeji A. Asuni, Benny Bang-Andersen, Wissam Beaino, Guus A. M. S. Dongen, Danielle J. Vugts
Journal: Theranostics (2022)

The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have shown that a better understanding of immunotherapy for the treatment of neurodegenerative diseases is needed. 89Zr-immuno-PET could be a suitable tool to open new avenues for the diagnosis of CNS disorders, monitoring disease progression, and assessment of novel therapeutics.Herein, three different 89 Zr-labeling strategies and direct radioiodination with 125 I of a bispecific anti-amyloid-beta aducanumab derivate, consisting of aducanumab with a C-terminal fused anti-transferrin receptor binding single chain Fab fragment derived from 8D3 (Adu-8D3), were compared ex vivo and in vivo with regard to brain uptake and target engagement in an APP/PS1 Alzheimer's disease mouse model and wild type animals.Methods: Adu-8D3 and a negative control antibody, based on the HIV specific B12 antibody also carrying C-terminal fused 8D3 scFab (B12-8D3), were each conjugated with NCS-DFO, NCS-DFO*, or TFP-N-suc-DFO-Fe-ester, followed by radiolabeling with 89 Zr. 125 I was used as a substitute for 124 I for labeling of both antibodies.30 µg of radiolabeled mAb, corresponding to approximately 6 MBq 89 Zr or 2.5 MBq 125 I, were injected per mouse.PET imaging was performed 1, 3 and 7 days post injection (p.i.).All mice were sacrificed on day 7 p.i. and subjected to ex vivo biodistribution and brain autoradiography.Immunostaining on brain tissue was performed after autoradiography for further validation.Results: Ex vivo biodistribution revealed that the brain uptake of [ 89 Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its 125 I-analog (2.21 ±0.15 %ID/g).[ 89 Zr]Zr-DFO-NCS-Adu-8D3 and [ 89 Zr]Zr-DFO-N-suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the 89 Zr-labeled controls (B12-8D3).Autoradiography of [ 89 Zr]Zr-DFO*-NCS-Adu-8D3 and [ 125 I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity.In contrast, the [ 89 Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for 89 Zr-immuno-PET imaging of the brain in comparison to DFO* for Adu-8D3.This was confirmed by day 7 PET images showing only amyloid-beta related brain uptake for [ 89 Zr]Zr-DFO*-NCS-Adu-8D3.In wild type animals, such an uptake was not observed.Immunostaining showed a co-localization of all administered Adu-8D3 conjugates with amyloid-beta plaques. Conclusion:We successfully demonstrated that 89 Zr-immuno-PET is suitable for imaging and quantifying amyloid-beta specific brain uptake using a bispecific aducanumab brain shuttling antibody, Adu-8D3, but only when using the novel chelator DFO*, and not DFO, for labeling with 89 Zr.